Methods of preventing platelet activation

ABSTRACT

A method of preventing or reducing the occurrence of malondiadehyde and/or levuglandin protein modification in a subject in need thereof, comprising administering to said subject an effective amount of at least one γ-KA scavenger compound, or a pharmaceutically acceptable salt thereof.

PRIOR APPLICATIONS

This application claims benefit to U.S. Application No. 62/270,435,filed Dec. 21, 2015, the contents of which are incorporated herein byreference in their entirety.

GOVERNMENT INTEREST

This invention was made with government support under grant numbersGM087603 and P50HL81009 awarded by the National Institutes of Health.The government has certain rights in the invention.

INTRODUCTION AND SUMMARY

Analysis of platelet activation is typically done on fresh blood becauseplatelets are very quickly activated after blood is drawn. Currentinhibitors of platelet activation used at time of phlebotomy do notsufficiently prevent platelet activation if the tubes of blood are notquickly used for analysis. It is therefore recommended in the field thatplatelet analysis be done within 2 hours post drawing. This timeconstraint restricts the ability to do large scale testing and tocentralize platelet analysis in a multicenter study. The presentinvention allows analysis, e.g., by flow cytometry, of plateletactivation of blood drawn up to about 72 hours before analysis.

The present invention is also directed to methods of prevention ofmalondialdehyde modification of platelet proteins. The thromboxanesynthase converts prostaglandin H₂ to thromboxane A₂ and malondialdehyde(MDA) in approximately equimolar amounts. A reactive dicarbonyl, MDAforms covalent adducts of amino groups, including the ε-amine of lysine.Utilizing a novel LC/MS/MS method for analysis of one of the MDAadducts, the dilysyl-MDA crosslink, the present inventors havedemonstrated that dilysyl-MDA crosslinks in human platelets are formedfollowing platelet activation via the COX-1/thromboxane synthasepathway. Compounds of the present invention were shown to react with MDApreferentially, thereby preventing formation of lysine adducts.Dilysyl-MDA crosslinks were measured in two diseases known to beassociated with increased platelet activation. Levels of plateletdilysyl-MDA crosslinks were increased by 2 fold in metabolic syndromerelative to healthy subjects, and by 1.9 fold in sickle cell disease. Inpatients with sickle cell disease, the reduction of platelet dilysyl-MDAcrosslinks following NSAID administration provided evidence that MDAmodifications of platelet proteins in this disease are derived from thecyclooxygenase pathway. In summary, MDA adducts of platelet proteinsthat crosslink lysines are formed on platelet activation and areincreased in diseases associated with platelet activation. These proteinmodifications can be prevented by salicylamine-related scavengers.

Thus, another aspect of the present invention is a method of preventingthe formation of malondialdehyde adducts. Another aspect of the presentinvention is a method of protecting histones and DNA from reactivedicarbonyls.

Another aspect of the present invention is the inhibition of plateletactivation.

Another aspect of the present invention is a method of preventing orreducing the occurrence of malondiadehyde and/or levuglandin proteinmodification in a subject in need thereof, comprising administering tosaid subject an effective amount of at least one γ-KA scavengercompound, or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention is a method of preventing theformation of malondiadehyde adducts, comprising administering to asubject in need thereof an effective malondiadehyde adduct reducingamount of a of at least one γ-KA scavenger compound, or apharmaceutically acceptable salt thereof.

Another aspect of the present invention is a method of inhibitingplatelet activation, comprising administering to a subject in needthereof an effective malondiadehyde adduct reducing amount of a of atleast one γ-KA scavenger compound, or a pharmaceutically acceptable saltthereof.

Another aspect of the present invention is a method of treating sicklecell anemia.

Another aspect of the present invention is a method of treatingmetabolic syndrome.

Another aspect of the present invention is method of treatingthrombosis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows LC/ESI/MS/MS spectra and selected ion monitoring ofdilysyl-MDA crosslink, and structure of product ions. The [MH]+ ions ofdilysyl-MDA crosslink (m/z 329) were subjected to CID. The product ionswere scanned from m/z 50 to 350. The spectra recorded at −15 (upperspectrum) and −28 eV (lower spectrum) are shown with the proposedstructures of the major fragment ions. The corresponding m/z for theinternal standard (IS) are presented in parenthesis.

FIG. 2 shows typical SRM chromatograms of the dilysyl-MDA crosslink.Dilysyl-MDA crosslinks were purified by Oasis cartridge and HPLC, afterbeing digested to single amino acid by step-digestion with proteases.The fractions containing the radioactivity were pooled, concentrated,and analyzed by LC/MS/MS. Selected reaction monitoring (SRM) of specifictransition ions for the precursor ions at m/z 329 (upper panels) or m/z341 (IS, lower panels), are shown. The IS was prepared from reaction ofMDA with lysine and is assumed to be crosslinked to the α-amine, thusyielding a shorter retention time.

FIG. 3 is a graph that shows dilysyl-MDA crosslinks are formed inactivated human washed platelets in a thromboxane synthase-dependentway. Human washed platelets were preincubated with vehicle (subject) oreither with 100 μM Ozagrel, 100 μM Aspirin or 1.5 mM SA or 3-MoSA for 40min. After incubation, 5 μM arachidonic acid was added for 2 h. (A)Dilysyl-MDA crosslinks were purified and quantified by isotopic dilutionusing LC/MS/MS. The product ions at m/z 294 for dilysyl-MDA crosslinkand m/z 306 for the IS were monitored. *p<0.0001 vs all otherconditions. (B) As a measure of COX activity, TxB₂ was determined byGC/MS from 50 μl of washed platelet. **p<0.0001 vs subject, Oz or ASA.One-way ANOVA followed by Tukey's multiple comparisons test was used.(n>6).

FIG. 4 are graphs that show lysine-MDA adducts formation is inhibited bySA or MoSA. Lysine and MDA were incubated at 37° C. for 4 h in presenceof vehicle (A), MoSA (B), SA (C) or 4-HoBA (D). The profiles weredetermined by HPLC as described in Methods section.

FIG. 5 is a graph that shows scavenger-MDA adducts are formed when humanwashed platelets are activated in presence of scavengers. Human washedplatelets were pre-incubated with MoSA or SA (1.5 mM finalconcentration) for 40 min, followed by arachidonic acid (at 5 μM finalconcentration) for an additional 2 h. Scavengers-MDA adducts wereextracted with ethyl acetate, and analyzed by LC/MS/MS by monitoring thespecific SRM transitions of m/z 208→136 (SA-MDA) and 178→106 (MoSA-MDA).+p<0.0001 vs subject or AA. One-way ANOVA followed by Tukey's multiplecomparisons test was used. (n>7).

FIG. 6 are graphs that show dilysyl-MDA crosslinks isolated from washedplatelets of patients with MetSyn or SCD. The dilysyl-MDA crosslink waspurified and analyzed by LC/MS/MS. (A) Levels of dilysyl-MDA crosslinkswere 2-fold higher in un-activated platelets from MetSyn patients (n=25)than healthy volunteers (n=20, p=0.0002 by t-test (two-tailed) withwelch's correction). (B) In platelets from SCD patients with no NSAIDs(n=12), levels of dilysyl-MDA crosslinks were 2-fold higher than fromhealthy volunteers (n=20, p<0.0001, SCD no NSAIDs vs healthy patients orvs SCD NSAIDs patients). One-way ANOVA followed by Tukey's multiplecomparisons test was used. The bars represent the mean for eachcategory. No statistical significance was found between Healthyvolunteers and SCD NSAIDs (n=9) patients.

FIG. 7 is a graph that shows inhibition of formation of LG adducts inplatelets for an embodiment of the present invention.

FIG. 8 is graph that shows the scavenger EtSA blocks LG-lysyl adductformation on histones.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document. The information provided in thisdocument, and particularly the specific details of the describedexemplary embodiments, is provided primarily for clearness ofunderstanding and no unnecessary limitations are to be understoodtherefrom. In case of conflict, the specification of this document,including definitions, will control.

While the terms used herein are believed to be well understood by thoseof ordinary skill in the art, certain definitions are set forth tofacilitate explanation of the presently-disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong.

Before the present compounds, compositions, articles, systems, devices,and/or methods are disclosed and described, it is to be understood thatthey are not limited to specific synthetic methods unless otherwisespecified, or to particular reagents unless otherwise specified, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only andis not intended to be limiting. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, example methods andmaterials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedherein can be different from the actual publication dates, which need tobe independently confirmed.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a functionalgroup,” “an alkyl,” or “a residue” includes mixtures of two or more suchfunctional groups, alkyls, or residues, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, a further aspect includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms a further aspect. It willbe further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance can or cannot occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the term “subject” refers to a target of administration.The subject of the herein disclosed methods can be a vertebrate, such asa mammal, a fish, a bird, a reptile, or an amphibian. Thus, the subjectof the herein disclosed methods can be a human, non-human primate,horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent.The term does not denote a particular age or sex. Thus, adult andnewborn subjects, as well as fetuses, whether male or female, areintended to be covered. A patient refers to a subject afflicted with adisease or disorder. The term “patient” includes human and veterinarysubjects.

As used herein, the term “treatment” refers to the medical management ofa patient with the intent to cure, ameliorate, stabilize, or prevent adisease, pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

As used herein, the term “prevent” or “preventing” refers to precluding,averting, obviating, forestalling, stopping, or hindering something fromhappening, especially by advance action. It is understood that wherereduce, inhibit or prevent are used herein, unless specificallyindicated otherwise, the use of the other two words is also expresslydisclosed. As can be seen herein, there is overlap in the definition oftreating and preventing.

As used herein, the term “diagnosed” means having been subjected to aphysical examination by a person of skill, for example, a physician, andfound to have a condition that can be diagnosed or treated by thecompounds, compositions, or methods disclosed herein. As used herein,the phrase “identified to be in need of treatment for a disorder,” orthe like, refers to selection of a subject based upon need for treatmentof the disorder. For example, a subject can be identified as having aneed for treatment of a disorder (e.g., a disorder related toinflammation) based upon an earlier diagnosis by a person of skill andthereafter subjected to treatment for the disorder. It is contemplatedthat the identification can, in one aspect, be performed by a persondifferent from the person making the diagnosis. It is also contemplated,in a further aspect, that the administration can be performed by one whosubsequently performed the administration.

As used herein, the terms “administering” and “administration” refer toany method of providing a pharmaceutical preparation to a subject. Suchmethods are well known to those skilled in the art and include, but arenot limited to, oral administration, transdermal administration,administration by inhalation, nasal administration, topicaladministration, intravaginal administration, ophthalmic administration,intraaural administration, intracerebral administration, rectaladministration, and parenteral administration, including injectable suchas intravenous administration, intra-arterial administration,intramuscular administration, and subcutaneous administration.Administration can be continuous or intermittent. In various aspects, apreparation can be administered therapeutically; that is, administeredto treat an existing disease or condition. In further various aspects, apreparation can be administered prophylactically; that is, administeredfor prevention of a disease or condition.

As used herein, the term “effective amount” refers to an amount that issufficient to achieve the desired result or to have an effect on anundesired condition. For example, a “therapeutically effective amount”refers to an amount that is sufficient to achieve the desiredtherapeutic result or to have an effect on undesired symptoms, but isgenerally insufficient to cause adverse side effects. The specifictherapeutically effective dose level for any particular patient willdepend upon a variety of factors including the disorder being treatedand the severity of the disorder; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration; the route of administration; the rate of excretion ofthe specific compound employed; the duration of the treatment; drugsused in combination or coincidental with the specific compound employedand like factors well known in the medical arts. For example, it is wellwithin the skill of the art to start doses of a compound at levels lowerthan those required to achieve the desired therapeutic effect and togradually increase the dosage until the desired effect is achieved. Ifdesired, the effective daily dose can be divided into multiple doses forpurposes of administration. Consequently, single dose compositions cancontain such amounts or submultiples thereof to make up the daily dose.The dosage can be adjusted by the individual physician in the event ofany contraindications. Dosage can vary, and can be administered in oneor more dose administrations daily, for one or several days. Guidancecan be found in the literature for appropriate dosages for given classesof pharmaceutical products. In further various aspects, a preparationcan be administered in a “prophylactically effective amount”; that is,an amount effective for prevention of a disease or condition.

As used herein, the term “pharmaceutically acceptable carrier” refers tosterile aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, as well as sterile powders for reconstitution into sterileinjectable solutions or dispersions just prior to use. Examples ofsuitable aqueous and nonaqueous carriers, diluents, solvents or vehiclesinclude water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol and the like), carboxymethylcellulose and suitablemixtures thereof, vegetable oils (such as olive oil) and injectableorganic esters such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants. These compositions can also contain adjuvantssuch as preservatives, wetting agents, emulsifying agents and dispersingagents. Prevention of the action of microorganisms can be ensured by theinclusion of various antibacterial and antifungal agents such asparaben, chlorobutanol, phenol, sorbic acid and the like. It can also bedesirable to include isotonic agents such as sugars, sodium chloride andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the inclusion of agents, such as aluminummonostearate and gelatin, which delay absorption. Injectable depot formsare made by forming microencapsule matrices of the drug in biodegradablepolymers such as polylactide-polyglycolide, poly(orthoesters) andpoly(anhydrides). Depending upon the ratio of drug to polymer and thenature of the particular polymer employed, the rate of drug release canbe controlled. Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues. The injectable formulations can be sterilized, forexample, by filtration through a bacterial-retaining filter or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable media just prior to use. Suitable inertcarriers can include sugars such as lactose. Desirably, at least 95% byweight of the particles of the active ingredient have an effectiveparticle size in the range of 0.01 to 10 micrometers.

As used herein, the term “scavenger” or “scavenging” refers to achemical substance that can be administered in order to remove orinactivate impurities or unwanted reaction products. For example, theisoketals irreversibly adduct specifically to lysine residues onproteins. The isoketal scavengers of the present invention react withisoketals before they adduct to the lysine residues. Accordingly, thecompounds of the present invention “scavenge” isoketals, therebypreventing them from adducting to proteins.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, and aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described below. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms, such as nitrogen, canhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. This disclosure is not intended to be limited in any mannerby the permissible substituents of organic compounds. Also, the terms“substitution” or “substituted with” include the implicit proviso thatsuch substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., a compound that does not spontaneouslyundergo transformation such as by rearrangement, cyclization,elimination, etc.

The term “alkyl” as used herein is a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl,isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl,dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. Thealkyl group can be cyclic or acyclic. The alkyl group can be branched orunbranched. The alkyl group can also be substituted or unsubstituted.For example, the alkyl group can be substituted with one or more groupsincluding, but not limited to, optionally substituted alkyl, cycloalkyl,alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, orthiol, as described herein. A “lower alkyl” group is an alkyl groupcontaining from one to six (e.g., from one to four) carbon atoms.

Throughout the specification “alkyl” is generally used to refer to bothunsubstituted alkyl groups and substituted alkyl groups; however,substituted alkyl groups are also specifically referred to herein byidentifying the specific substituent(s) on the alkyl group. For example,the term “halogenated alkyl” specifically refers to an alkyl group thatis substituted with one or more halide, e.g., fluorine, chlorine,bromine, or iodine. The term “alkoxyalkyl” specifically refers to analkyl group that is substituted with one or more alkoxy groups, asdescribed below. The term “alkylamino” specifically refers to an alkylgroup that is substituted with one or more amino groups, as describedbelow, and the like. When “alkyl” is used in one instance and a specificterm such as “alkylalcohol” is used in another, it is not meant to implythat the term “alkyl” does not also refer to specific terms such as“alkylalcohol” and the like.

This practice is also used for other groups described herein. That is,while a term such as “cycloalkyl” refers to both unsubstituted andsubstituted cycloalkyl moieties, the substituted moieties can, inaddition, be specifically identified herein; for example, a particularsubstituted cycloalkyl can be referred to as, e.g., an“alkylcycloalkyl.” Similarly, a substituted alkoxy can be specificallyreferred to as, e.g., a “halogenated alkoxy,” a particular substitutedalkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, thepractice of using a general term, such as “cycloalkyl,” and a specificterm, such as “alkylcycloalkyl,” is not meant to imply that the generalterm does not also include the specific term.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is atype of cycloalkyl group as defined above, and is included within themeaning of the term “cycloalkyl,” where at least one of the carbon atomsof the ring is replaced with a heteroatom such as, but not limited to,nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group andheterocycloalkyl group can be substituted or unsubstituted. Thecycloalkyl group and heterocycloalkyl group can be substituted with oneor more groups including, but not limited to, optionally substitutedalkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl,sulfo-oxo, or thiol as described herein.

The term “polyalkylene group” as used herein is a group having two ormore CH₂ groups linked to one another. The polyalkylene group can berepresented by a formula —(CH₂)_(a)—, where “a” is an integer of from 2to 500.

The terms “alkoxy” and “alkoxyl” as used herein to refer to an alkyl orcycloalkyl group bonded through an ether linkage; that is, an “alkoxy”group can be defined as —OA¹ where A¹ is alkyl or cycloalkyl as definedabove. “Alkoxy” also includes polymers of alkoxy groups as justdescribed; that is, an alkoxy can be a polyether such as —OA¹-OA² or—OA¹-(OA²)_(a)-OA³, where “a” is an integer of from 1 to 200 and A¹, A²,and A³ are alkyl and/or cycloalkyl groups.

The terms “amine” or “amino” as used herein are represented by a formulaNA¹A²A³, where A¹, A², and A³ can be, independently, hydrogen oroptionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

The term “hydroxyl” as used herein is represented by a formula —OH.

The term “nitro” as used herein is represented by a formula —NO₂.

The term “pharmaceutically acceptable” describes a material that is notbiologically or otherwise undesirable, i.e., without causing anunacceptable level of undesirable biological effects or interacting in adeleterious manner.

Examples of compounds of the present invention include, but are notlimited to, compounds selected from the formula:

wherein:R is N or C;R₂ is independently H, substituted or unsubstituted alkyl;R₃ is H, halogen, alkoxy, hydroxyl, nitro;R₄ is H, substituted or unsubstituted alkyl, carboxyl; andpharmaceutically acceptable salts thereof.

The compound may be chosen from:

or a pharmaceutically acceptable salt thereof.

The compound may also be chosen from:

or a pharmaceutically acceptable salt thereof.

The compounds or analogs may also be chosen from:

or a pharmaceutically acceptable salt thereof.

The compounds may also be chosen from:

or a pharmaceutically acceptable salt thereof.

The compounds may also be chosen from

or a pharmaceutically acceptable salt thereof.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts prepared from pharmaceutically acceptable non-toxic bases oracids. When the compound of the present invention is acidic, itscorresponding salt can be conveniently prepared from pharmaceuticallyacceptable non-toxic bases, including inorganic bases and organic bases.Salts derived from such inorganic bases include aluminum, ammonium,calcium, copper (-ic and -ous), ferric, ferrous, lithium, magnesium,manganese (-ic and -ous), potassium, sodium, zinc and the like salts.Particularly preferred are the ammonium, calcium, magnesium, potassiumand sodium salts. Salts derived from pharmaceutically acceptable organicnon-toxic bases include salts of primary, secondary, and tertiaryamines, as well as cyclic amines and substituted amines such asnaturally occurring and synthesized substituted amines. Otherpharmaceutically acceptable organic non-toxic bases from which salts canbe formed include ion exchange resins such as, for example, arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like.

Embodiments of the present invention also include a compositioncomprising a mixture of components, which the present inventorsdiscovered to be useful for platelet stabilization or prevention ofplatelet activation of drawn blood. The beneficial features of thiscomposition allow platelet analysis of the drawn blood to be conductedmore than 2 hours post-draw and in some embodiments, up to about 72hours post-draw. The composition, kits, and methods of thepresently-disclosed subject matter can be used to “fix” the platelets inthe activation state in which they were at the time of blood drawing.

In some embodiments, the composition includes an irreversible inhibitorof COX-1 together with a series of compounds for limiting orsubstantially preventing activation of the reinforcement pathway ofplatelets, including a compound for blocking activation of thepurinergic receptors, a compound for blocking the thromboxane receptor,a compound for increasing intracellular cAMP concentration, and acompound that blocks potassium-dependent coagulation factors. In someembodiments, the composition further includes a compound that is areactive aldehyde scavenger that protects platelet proteins fromcovalent modification by dicarbonyls, which are also products of COX-1activity and oxidative stress.

In some embodiments, the composition includes EDTA (blockspotassium-dependent coagulation factors), aspirin (COX-1 inhibitor),apyrase (blocks activation of the purinergic receptors),carbaprostacyclin (increases intracellular cAMP concentration), and SQ29,548 (blocks the thromboxane receptor). In some embodiments, thecomposition further includes pyridoxamine (aldehyde scavenger). In someembodiments, for each about 5 ml of blood, the composition includes EDTA(about 9 mg), aspirin (100 μM), apyrase (2 Units), carbaprostacyclin (1μM), SQ 29,548 (10 nM) and pyridoxamine (1 mM) final concentration.

The presently-disclosed subject matter further includes kits. The kitscan include a phlebotomy tube, vacutainer tube, syringe, or other devicefor collecting blood, packaged together with the composition of thepresently disclosed subject matter. In some embodiments, the compositionis contained within the device for collecting blood.

The presently-disclosed subject matter further includes methods. In someembodiments, the method involves contacting collected blood with anamount of the composition that is effective for substantially preventingor reducing platelet activation. In some embodiments, the methodinvolves collecting blood using a device that contains the composition.

In some embodiments of the presently-disclosed subject matter, blood iscollected into a 5 ml syringe containing 9 mg EDTA, aspirin (100 μM),apyrase (2 Units), carbaprostacyclin (1 μM), SQ 29,548 (10 nM) andpyridoxamine (1 mM) final concentration, to prevent ex vivo plateletactivation. The blood is incubated at room temperature for 40 min.Following this incubation, blood can be stored at 4° C. or shipped incoldpack to the place where the analyses are done.

For purposes of the presently-disclosed subject matter, prevention ofplatelet activation can be assessed by measuring expression ofP-selectin at a time-point of interest, e.g., 72 hours, post-draw.Efficacy can be tested using markers of platelet activation, includingmalondialdehyde adducts on proteins by mass spectrometry and plateletexpression of P-selectin by flow cytometry. Other markers of plateletactivation include, activated GPIIb3a, platelet-monocyte aggregates andplatelet-monocyte aggregate-associated P-selectin expression.

The present inventors have demonstrated that platelet activation leadsto formation of MDA adducts of platelet proteins via theCOX-1/thromboxane synthase pathway. Metabolism of PGH₂ by thethromboxane synthase yields MDA in amounts approximately equivalent tothromboxane A₂ (1). As a reactive 1,3-dicarbonyl, MDA forms adducts ofproteins, such as the N^(ε)-propenal-lysine, that can further react withan additional amino group to form intra- and inter-molecular crosslinks.

Evidence that MDA adducts of platelet proteins are formed duringactivation of platelets ex vivo engendered the hypothesis that thesemodifications of platelet proteins also would occur in diseases in whichplatelet activation occurs in vivo. Development of an LC/MS/MS methodfor analysis of one of these adducts (the dilysyl-MDA crosslink) hasmade it possible to address this hypothesis.

Platelet function is disordered in MetSyn. Platelets from these patientsare hyperactive when studied ex vivo (14-16), and there is evidence forin vivo platelet activation (14, 15, 17-19). The present inventors havefound that dilysyl-MDA crosslinks are elevated in MetSyn to a mean level2-fold greater than normal (p=0.0002), and 52% of the patients hadvalues greater than the upper limit of the normal range. Plateletactivation also is a feature of SCD, and levels of dilysyl-MDAcrosslinks also are increased in platelets from patients with thisdisease (p<0.0001). Thus, increased levels of MDA adducts of plateletproteins are correlated with diseases that produce platelet activation,recapitulating the formation of these adducts during ex vivo plateletactivation. These findings show that analysis of dilysyl-MDA crosslinkscould provide biomarker and therapeutic options for in vivo plateletdysfunction.

The origin of the MDA adducts from the platelet COX-1/thromboxanesynthase pathway is clearly demonstrated in the experiments with normalplatelets activated ex vivo, in which aspirin and the thromboxanesynthase inhibitor, ozagrel, reduce the dilysyl-MDA crosslinks to thelevels in non-activated platelets. A major contribution of the COX-1pathway to the increased formation of dilysyl-MDA crosslinks SCD also issuggested by the finding that therapeutic use of the cyclooxygenaseinhibitor, ketorolac, in these patients was associated with a reductionof dilysyl-MDA crosslinks to the level found in normal platelets; thisoccurred in conjunction with a 69% reduction in serum TxB₂. However, MDAalso is a product of radical catalyzed lipid peroxidation and reactiveoxygen species are generated by platelet activation (20). Whereas normalplatelets have robust anti-oxidant defenses and virtually all of MDAadduct formation is COX-1 derived, oxidant stress is evident inplatelets in MetSyn. The present inventors have found an increase inF₂-isoprostanes esterified to platelet lipids in MetSyn, and ascorbatelevels are decreased (21, 22). Thus, it is possible that in diseasessuch as MetSyn, platelet activation could generate MDA via radicalcatalyzed lipid peroxidation in addition to that generated via COX-1. Ifthat were the case in MetSyn, the in vivo formation of MDA-proteinadducts as a marker of platelet dysfunction would be amplified.

These findings provide a basis for considering the dilysyl-MDA crosslinkas a biomarker of in vivo platelet dysfunction in MetSyn. Thealternative approach to assessing platelet activation-induced metabolismof arachidonic acid via the COX-1/thromboxane synthase pathway is basedon our discovery that 11-dehydrothromboxane B₂ (TxM) is a majormetabolite of TxB₂ in humans (23). An average of 70-80% of urinary TxMis derived from platelets (24), but the amount from non-platelet sourcesis highly variable (10, 24, 25). Thus, the urinary excretion of TxM hasbeen a useful indicator of large increases in platelet activation suchas occurs in the acute coronary syndrome (26) and the anti-phospholipidantibody syndrome (27, 28). However, to assess individual changes inurinary TxM closer to the normal range is problematic because theamounts derived from non-platelet sources is variable; for example,non-platelet production of TxB₂ is increased in smokers (25). Bycontrast, formation of MDA specifically in the platelet is the likelysource of virtually all of the dilysyl-MDA crosslinks in platelets.

Compound of the present invention, including SA and its analogues3-MoSA, EtSA and MeSA prevent formation of MDA adducts of plateletproteins. Demonstration that MDA reacts preferentially with thesescavengers to form covalent adducts of the scavengers provides amechanism for their ability to protect proteins from the attack of MDA.In addition to the COX-1 dependent formation of the 1,3-dicarbonyl, MDA,upon platelet activation, platelet COX-1 also is the source of highlyreactive 1,4-dicarbonyls, the levuglandins, which are formed fromrearrangement of PGH₂. The present inventors previously have shown thatSA also reacts with the levuglandins to prevent formation oflevuglandinyl adducts of platelet proteins (4). Thus, these2-hydroxybenzylamines are highly reactive with both 1,3- and1,4-dicarbonyls, acting to scavenge these and similar dicarbonyls toprevent them from reacting with amino groups of cellular molecules. Theefficacy of these scavengers to prevent protein modification by MDA andlevuglandins in the platelet can be extended to other cells and tolipoproteins.

Accordingly, one embodiment of the present invention is a method ofpreventing or reducing the occurrence of malondiadehyde and/orlevuglandin protein modification in a subject in need thereof,comprising administering to said subject an effective amount of at leastone γ-KA scavenger compound, or a pharmaceutically acceptable saltthereof.

Another embodiment of the present invention is a method of preventingthe formation of malondiadehyde adducts, comprising administering to asubject in need thereof an effective malondiadehyde adduct reducingamount of a of at least one γ-KA scavenger compound, or apharmaceutically acceptable salt thereof.

Another embodiment of the present invention is a method of inhibitingplatelet activation, comprising administering to a subject in needthereof an effective malondiadehyde adduct reducing amount of a of atleast one γ-KA scavenger compound, or a pharmaceutically acceptable saltthereof.

In aspects of the invention, the subject has or is at greater risk thanthe general population for a disease or condition selected from thegroup consisting of sickle cell anemia, metabolic syndrome, thrombosis.The thrombosis may be venous thrombosis, deep vein thrombosis, portalvein thrombosis, renal vein thrombosis, arterial thrombosis, andpost-surgical thrombotic complications arising from angioplasty andorgan transplantation.

In certain aspects of the above methods, the compound is salicylamine.In other embodiments, the compound is selected from the followingformula:

wherein: R is N or C; R₂ is independently H, substituted orunsubstituted alkyl; R₃ is H, halogen, alkoxy, hydroxyl, nitro; R₄ is H,substituted or unsubstituted alkyl, carboxyl; and pharmaceuticallyacceptable salts thereof.

In other embodiments, the compound is selected from the followingformula:

or a pharmaceutically acceptable salt thereof.

In other embodiments, the compound is selected from the followingformula:

or a pharmaceutically acceptable salt thereof.

In other embodiments, the compound is selected from the followingformula:

or a pharmaceutically acceptable salt thereof.

In one aspect, the invention relates to pharmaceutical compositionscomprising the disclosed compounds. That is, a pharmaceuticalcomposition can be provided comprising a therapeutically effectiveamount of at least one disclosed compound or at least one product of adisclosed method and a pharmaceutically acceptable carrier.

In certain aspects, the disclosed pharmaceutical compositions comprisethe disclosed compounds (including pharmaceutically acceptable salt(s)thereof) as an active ingredient, a pharmaceutically acceptable carrier,and, optionally, other therapeutic ingredients or adjuvants. The instantcompositions include those suitable for oral, rectal, topical, andparenteral (including subcutaneous, intramuscular, and intravenous)administration, although the most suitable route in any given case willdepend on the particular host, and nature and severity of the conditionsfor which the active ingredient is being administered. Thepharmaceutical compositions can be conveniently presented in unit dosageform and prepared by any of the methods well known in the art ofpharmacy.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts prepared from pharmaceutically acceptable non-toxic bases oracids. When the compound of the present invention is acidic, itscorresponding salt can be conveniently prepared from pharmaceuticallyacceptable non-toxic bases, including inorganic bases and organic bases.Salts derived from such inorganic bases include aluminum, ammonium,calcium, copper (-ic and -ous), ferric, ferrous, lithium, magnesium,manganese (-ic and -ous), potassium, sodium, zinc and the like salts.Particularly preferred are the ammonium, calcium, magnesium, potassiumand sodium salts. Salts derived from pharmaceutically acceptable organicnon-toxic bases include salts of primary, secondary, and tertiaryamines, as well as cyclic amines and substituted amines such asnaturally occurring and synthesized substituted amines. Otherpharmaceutically acceptable organic non-toxic bases from which salts canbe formed include ion exchange resins such as, for example, arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like.

As used herein, the term “pharmaceutically acceptable non-toxic acids”includes inorganic acids, organic acids, and salts prepared therefrom,for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric,isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic,nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric,p-toluenesulfonic acid and the like. Preferred are citric, hydrobromic,hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

In practice, the compounds of the invention, or pharmaceuticallyacceptable salts thereof, of this invention can be combined as theactive ingredient in intimate admixture with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques. Thecarrier can take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., oral or parenteral(including intravenous). Thus, the pharmaceutical compositions of thepresent invention can be presented as discrete units suitable for oraladministration such as capsules, cachets or tablets each containing apredetermined amount of the active ingredient. Further, the compositionscan be presented as a powder, as granules, as a solution, as asuspension in an aqueous liquid, as a non-aqueous liquid, as anoil-in-water emulsion or as a water-in-oil liquid emulsion. In additionto the common dosage forms set out above, the compounds of theinvention, and/or pharmaceutically acceptable salt(s) thereof, can alsobe administered by controlled release means and/or delivery devices. Thecompositions can be prepared by any of the methods of pharmacy. Ingeneral, such methods include a step of bringing into association theactive ingredient with the carrier that constitutes one or morenecessary ingredients. In general, the compositions are prepared byuniformly and intimately admixing the active ingredient with liquidcarriers or finely divided solid carriers or both. The product can thenbe conveniently shaped into the desired presentation.

Thus, the pharmaceutical compositions of this invention can include apharmaceutically acceptable carrier and a compound or a pharmaceuticallyacceptable salt of the compounds of the invention. The compounds of theinvention, or pharmaceutically acceptable salts thereof, can also beincluded in pharmaceutical compositions in combination with one or moreother therapeutically active compounds. The pharmaceutical carrieremployed can be, for example, a solid, liquid, or gas. Examples of solidcarriers include lactose, terra alba, sucrose, talc, gelatin, agar,pectin, acacia, magnesium stearate, and stearic acid. Examples of liquidcarriers are sugar syrup, peanut oil, olive oil, and water. Examples ofgaseous carriers include carbon dioxide and nitrogen.

In preparing the compositions for oral dosage form, any convenientpharmaceutical media can be employed. For example, water, glycols, oils,alcohols, flavoring agents, preservatives, coloring agents and the likecan be used to form oral liquid preparations such as suspensions,elixirs and solutions; while carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents, and the like can be used to form oralsolid preparations such as powders, capsules and tablets. Because oftheir ease of administration, tablets and capsules are the preferredoral dosage units whereby solid pharmaceutical carriers are employed.Optionally, tablets can be coated by standard aqueous or nonaqueoustechniques

A tablet containing the composition of this invention can be prepared bycompression or molding, optionally with one or more accessoryingredients or adjuvants. Compressed tablets can be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets can be made by molding in a suitable machine, a mixtureof the powdered compound moistened with an inert liquid diluent.

The pharmaceutical compositions of the present invention can comprise acompound of the invention (or pharmaceutically acceptable salts thereof)as an active ingredient, a pharmaceutically acceptable carrier, andoptionally one or more additional therapeutic agents or adjuvants. Theinstant compositions include compositions suitable for oral, rectal,topical, and parenteral (including subcutaneous, intramuscular, andintravenous) administration, although the most suitable route in anygiven case will depend on the particular host, and nature and severityof the conditions for which the active ingredient is being administered.The pharmaceutical compositions can be conveniently presented in unitdosage form and prepared by any of the methods well known in the art ofpharmacy.

Pharmaceutical compositions of the present invention suitable forparenteral administration can be prepared as solutions or suspensions ofthe active compounds in water. A suitable surfactant can be includedsuch as, for example, hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofin oils. Further, a preservative can be included to prevent thedetrimental growth of microorganisms.

Pharmaceutical compositions of the present invention suitable forinjectable use include sterile aqueous solutions or dispersions.Furthermore, the compositions can be in the form of sterile powders forthe extemporaneous preparation of such sterile injectable solutions ordispersions. In all cases, the final injectable form must be sterile andmust be effectively fluid for easy syringability. The pharmaceuticalcompositions must be stable under the conditions of manufacture andstorage; thus, preferably should be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol and liquid polyethyleneglycol), vegetable oils, and suitable mixtures thereof.

Pharmaceutical compositions of the present invention can be in a formsuitable for topical use such as, for example, an aerosol, cream,ointment, lotion, dusting powder, mouth washes, gargles, and the like.Further, the compositions can be in a form suitable for use intransdermal devices. These formulations can be prepared, utilizing acompound of the invention, or pharmaceutically acceptable salts thereof,via conventional processing methods. As an example, a cream or ointmentis prepared by mixing hydrophilic material and water, together withabout 5 wt % to about 10 wt % of the compound, to produce a cream orointment having a desired consistency.

Pharmaceutical compositions of this invention can be in a form suitablefor rectal administration wherein the carrier is a solid. It ispreferable that the mixture forms unit dose suppositories. Suitablecarriers include cocoa butter and other materials commonly used in theart. The suppositories can be conveniently formed by first admixing thecomposition with the softened or melted carrier(s) followed by chillingand shaping in molds.

In addition to the aforementioned carrier ingredients, thepharmaceutical formulations described above can include, as appropriate,one or more additional carrier ingredients such as diluents, buffers,flavoring agents, binders, surface-active agents, thickeners,lubricants, preservatives (including anti-oxidants) and the like.Furthermore, other adjuvants can be included to render the formulationisotonic with the blood of the intended recipient. Compositionscontaining a compound of the invention, and/or pharmaceuticallyacceptable salts thereof, can also be prepared in powder or liquidconcentrate form.

The compounds of the present invention can be administered as the soleactive pharmaceutical agent, or can be used in combination with one ormore other agents useful for treating or preventing variouscomplications, such as, for example, inflammation and otherinflammation-related diseases. When administered as a combination, thetherapeutic agents can be formulated as separate compositions that aregiven at the same time or different times, or the therapeutic agents canbe given as a single composition.

As indicated herein, the compounds of the present invention may be madeup in a solid form (including granules, powders or suppositories) or ina liquid form (e.g., solutions, suspensions, or emulsions). They may beapplied in a variety of solutions and may be subjected to conventionalpharmaceutical operations such as sterilization and/or may containconventional adjuvants, such as preservatives, stabilizers, wettingagents, emulsifiers, buffers etc.

Thus, for administration, the compounds of the present invention areordinarily combined with one or more adjuvants appropriate for theindicated route of administration. For example, they may be admixed withlactose, sucrose, starch powder, cellulose esters of alkanoic acids,stearic acid, talc, magnesium stearate, magnesium oxide, sodium andcalcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodiumalginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tabletedor encapsulated for conventional administration. Alternatively, they maybe dissolved in saline, water, polyethylene glycol, propylene glycol,carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanutoil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.Other adjuvants and modes of administration are well known in thepharmaceutical art. The carrier or diluent may include time delaymaterial, such as glyceryl monostearate or glyceryl distearate alone orwith a wax, or other materials well known in the art.

In therapeutic applications, the compounds of the present invention maybe administered to a mammalian patient in an amount sufficient to reduceor inhibit the desired indication. Amounts effective for this use dependon factors including, but not limited to, the route of administration,the stage and severity of the indication, the general state of health ofthe mammal, and the judgment of the prescribing physician. The compoundsof the present invention are safe and effective over a wide dosagerange. However, it will be understood that the amounts of pyridoxamineactually administered will be determined by a physician, in the light ofthe above relevant circumstances.

Pharmaceutically acceptable acid addition salts of the compoundssuitable for use in methods of the invention include salts derived fromnontoxic inorganic acids such as hydrochloric, nitric, phosphoric,sulfuric, hydrobromic, hydriodic, hydrofluoric, phosphorous, and thelike, as well as the salts derived from nontoxic organic acids, such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids,aliphatic and aromatic sulfonic acids, etc. Such salts thus includesulfate, pyrosulfate, bisulfate, sulfite, bisulfate, nitrate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, trifluoroacetate,propionate, caprylate, isobutyrate, oxalate, malonate, succinate,suberate, sebacate, fumarate, maleate, mandelate, benzoate,chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate,benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate,maleate, tartrate, methanesulfonate, and the like. Also contemplated aresalts of amino acids such as arginate and the like and gluconate,galacturonate, n-methyl glutamine, etc. (see, e.g., Berge et al., J.Pharmaceutical Science, 66: 1-19 (1977).

The acid addition salts of the basic compounds are prepared bycontacting the free base form with a sufficient amount of the desiredacid to produce the salt in the conventional manner. The free base formmay be regenerated by contacting the salt form with a base and isolatingthe free base in the conventional manner. The free base forms differfrom their respective salt forms somewhat in certain physical propertiessuch as solubility in polar solvents, but otherwise the salts areequivalent to their respective free base for purposes of the presentinvention.

EXAMPLES

The following example discusses MDA and dicarbonyl scavengers.

Activation of platelets signals cytosolic phospholipase A₂a activation,and an explosive release of arachidonic acid, which is metabolized bycyclooxygenase-1 (COX-1) to prostaglandin H₂ (PGH₂). In platelets, thethromboxane synthase enzyme catalyzes conversion of PGH₂ to boththromboxane A₂ and malondialdehyde (MDA) in approximately equimolaramounts (1).

MDA is an electrophile that reacts with amino groups, including theε-amine of protein lysines. Reaction of MDA with lysine in vitro leadsto formation of adducts with several structures (2, 3), including onethat results from the reaction of this dicarbonyl with two lysines toproduce intra- and inter-molecular crosslinks of macromolecules. Suchcrosslinks have been demonstrated when MDA is added to purified apoA-1in vitro (2).

Without being bound by theory or mechanism, this evidence indicates thatMDA is a major product of the thromboxane synthase and can modifyprotein structure in vitro indicates that platelet activation could leadto modification of platelet proteins by MDA.

Embodiments of the present invention demonstrate that activation ofplatelets ex vivo leads to thromboxane synthase-dependent MDAmodification of platelet proteins. A stable isotope dilution method foranalysis for the dilysyl-MDA crosslink utilizing liquid chromatographymass spectrometry (LC/MS/MS) has been developed, making it possible todemonstrate increased levels of MDA adducts of platelet proteins indiseases that are associated with increased platelet activation.

Methods

Chemicals and Reagents.

Ozagrel was from Cayman chemical Company (Ann Arbor, Mich.). TMP(precursor of MDA), arachidonic acid, sodium citrate, citric acid,lysine and aspirin (acetylsalicylic acid) were from Sigma-Aldrich (St.Louis, Mo.). Oasis™ HLB 1 cc cartridges containing 30 mg ofpoly(divinylbenzene-co-N-vinylpyrrolidone) copolymer, were obtained fromWaters Corp. (Milford, Mass.). Lysine-[³H, 99%] was from AmericanRadiolabeled Chemicals, Inc. (ARC, St Louis, Mo.) and lysine-[¹³C₆, 99%]was from Cambridge Isotope Laboratories, Inc. Salicylamine acetate (SA),3-methoxysalicylamine acetate (3-MoSA), 5-ethylsalicylamine (EtSA),5-methylsalicylamine (MeSA) and 4-hydroxybenzylamine (4-HoBA) weresynthesized as previously described (4). Phosphate buffered saline (10×)was from Fisher BioReagents-Fisher Scientific (Fair Lawn, N.J.). Spin Xcentrifuge filters were purchased from Costar (Corning, N.Y.). Theconjugated monoclonal antibodies anti-CD62p (P-selectin) andPE-conjugated anti-CD-41 were available from BD-Pharmingen (FranklinLakes, N.J.). All solvents were HPLC-grade.

Equipment.

A scintillation counter (liquid scintillation analyzer) Tri-Carb 1900TRwas from PerkinElmer. HPLC was performed using a Perkin Elmer Series 200system (Perkin Elmer Shelton, Conn.) consisting of a system controller,a LC-pump, an autosampler, a column oven, a degasser and a UV-visdetector. The analytical Aquasil C18 reverse phase column (250×4.6 mmi.d.), from Thermo scientific Chromatography (Pittsburgh, Pa.) or aPhenomenex Kinetex 2.6 μM XB-C18 100 A (75×2.1 mm i.d.), fromPhenomenex, (Torrance Calif.) were used. A Liquid Chromatography (LC)system was connected to a Finnigan TSQ Quantum Triple Quadrupole massspectrometer (Thermo Electron Inc., (San Jose, Calif.)) equipped with anelectrospray source. LC/MS/MS Xcalibur software (version 1.3;ThermoFinnigan) was used to operate the instrument and to process thedata. Aliquots of the samples were analyzed in the positive ion modeusing an YMC™-ODS-AQ (250×2.0 mm, 5 μm particle size) column from YMCCo., Ltd, (Allentown, Pa.).

Dilysyl-MDA Crosslinks Adducts.

For the characterization of dilysyl-MDA crosslinks, a mixture of [¹²C]lysine or [¹³C₆] lysine and TMP (ratio 3:2) was incubated in 1 N HCl for2 h at 37° C. The reaction was then neutralized and let stand overnightat room temperature. The dilysyl-MDA crosslinks were purified initiallywith solid phase extraction (SPE) columns (Oasis HLB cartridge),preconditioned with 2 ml of methanol and 2 ml of water. The samples wereloaded and then washed twice with 2 ml of water. The dilysyl-MDAcrosslink were eluted with (1:1) methanol/ethyl acetate. The cartridgerecovery was 99.83±16.58%. Under nitrogen, the residual product wasconcentrated down to 100 μl under nitrogen stream and analyzed byLC/MS/MS in the positive ion mode as described below. For determinationof product ions, collision-induced dissociation of molecular ions ofputative dilysyl-MDA crosslink was performed from −10 to −45 eV andscanning product ions from m/z 50 to 350. Spectra shown were obtained at−15 eV and −28 eV (FIG. 1).

An internal standard of dilysyl-MDA crosslink (IS) was prepared asdescribe above by reaction of TMP in the presence of [¹³C₆] lysine and[³H] lysine. The dilysyl-MDA crosslinks was extracted as describedpreviously and purified by HPLC. Fractions were collected every 0.5 minand presence of radioactivity was determined using a scintillationcounter. The fractions containing radioactive dilysyl-MDA crosslinks([³H] lysine tracer in internal standard) were combined and analyzed byLC/MS/MS. To achieve a higher peak intensity and better peak shape,optimization was done using a gradient of acetonitrile/water with anaddition of 0.1% formic acid to the mobile phase. The development of thechromatographic system was focused on shortening retention times whilepreserving the chromatographic separation of the analytes from thematrix contaminants. This was achieved by using a flow rate of 0.2ml/min with the gradient described in the Methods section. These ionshad an acceptable retention time (6.9 min and 6.7 min, respectively;FIG. 2). The overall analysis time was 18 min including re-equilibrationtime for the column with the initial mobile phase.

The radioactivity of the solution was counted using a scintillationcounter and the concentration was calculated from the specific activityof the [³H] lysine. The dilysyl-MDA crosslink is stable: 99.23% wasrecovered after storage up to 9 months at −80° C.

Method Validation Procedure.

Quality Control (QC) samples were prepared from washed plateletsobtained from aphorised platelet units obtained from the VanderbiltBlood bank. Aliquots of 3 mg of protein and all stock of dilysyl-MDAcrosslink standard were stored at −80° C. until used. The stability ofdilysyl-MDA crosslinks was assessed by analyzing samples stored at −80°C. for up to 9 months. Carry over was evaluated by placing vials ofblank solvent at several locations in the analysis set. Specificity andselectivity of the assay were assessed by comparing un-activated washedplatelet samples to washed platelet samples activated with 5 μMarachidonic acid for 2 h.

To determine the overall recovery of the cartridge, digested QC samplewas spiked with IS before or after SPE. The samples were purified andanalyzed. The peak areas obtained in neat solution standards as A, thecorresponding peak areas for post-extraction spiked samples as B, andthe peak areas for extracted samples as C, were used to calculated theIIS (ionization suppression) and AE (absolute recovery) values asfollow: IIS (%)=B/A×100%; AE (%)=CB×100% (n=3). The variability of thedigestion was assessed by comparing the amount of dilysyl-MDAcrosslinks, in samples split before or after digestion, and it wasdetermined according to the following equation: (% digestion=(amount ofcrosslinks in sample digested after split/amount of crosslinks in sampledigested before split)×100.

In order to assess the intra- and inter-batch precision and accuracy ofthe assay, QC samples at 1.5, 2.1, 5.8 and 12.5 mg were analyzed. Theintra-batch precision (repeatability) of the assay was evaluated by therelative standard deviation (% RSD) of three replicates and theinter-batch precision (reproducibility) was evaluated by the analysis ofstandard curve samples in three batches (in duplicate); %RSD=(SD/mean)×100. The assay was considered to be acceptable if %RSD<15%.

To determine the linearity of the assay, we prepared 1.8 mM stocksolution of dilysyl-MDA crosslink in water containing 0.1% formic acid.A dose-response curve ranging from 0.008 pg to 7.5 pg was prepared byweighing different amounts of synthetic dilysyl-MDA crosslink, and thearea under the curve (AUC) was recorded by LC/MS/MS as described inMethods section. Increasing amounts of dilysyl-MDA crosslinks gave alinear response with y=3.19e+006 x+442297, where y is the measured AUCof dilysyl-MDA crosslink and x is the weighed dilysyl-MDA crosslink inpg. The 1/slope and the correlation coefficient (r²) of the calibrationplot were 3.12e-007 and 0.93, respectively.

The limit of quantification (LOQ) and limit of detection (LOD) werecalculated directly from the calibration plot. The calibration samplewas prepared with [′²C] lysine, trace of [³H] lysine and TMP. Thereaction mixture was extracted by SPE as previously described in thesection of the preparation of dilysyl-MDA Crosslink standard. Theresponses AUC (area under curve) of the selected fragment relative tothe corresponding amount of adduct (in pg) were used to constructstandard curves by least square linear regression analysis. LOQ and LODwere calculated as 10σ/S and 3.3 σ/S, respectively, where a is thestandard deviation of y-intercept of regression equation and S is theslope of the calibration plot (5).

Sample Collection.

Written informed consent was obtained from study participants and humanblood was obtained following a protocol approved by the InstitutionalReview Board of Vanderbilt University. Samples were obtained from threedifferent populations: healthy subjects who had no component of theMetSyn or SCD, patients with MetSyn and patients with SCD. Patients withMetSyn were 35-75 year old and fit the MetSyn criteria in accord withthe American Heart Association/National Heart, Lung, and Blood Institutecriteria (6). Patients with SCD were 25-50 year old and were homozygoussickle (SS) or compound heterozygote with beta-thalassemia (Sβ-thal)phenotype. All recruited patients were queried about their NSAID use andenrolled only if they could confirm lack of NSAIDs use for at least 7days prior to sample collection.

Preparation of Washed Platelets.

Washed platelets were prepared following two different protocolsdepending on whether they were used for the in vitro studies of themechanism by which MDA adducts are generated, or for the in vivomeasurements of Protease-Derived Lysyl-MDA-Lysyl crosslink (dilysyl-MDAcrosslink) levels as a marker of platelet activation. The two protocolsare described below.

In Vitro Platelet Activation:

Blood obtained from healthy volunteers was collected into vacuum tubescontaining citrate, from a peripheral vein using a 21-gauge needle, andwashed platelets were prepared as previously described (7). Theplatelets were counted with a Coulter counter and diluted withresuspension buffer to a final concentration of 300,000 platelets/μl,and aliquoted in volumes corresponding to 500 μl. The aliquots werepreincubated for 40 min at room temperature with vehicle, thethromboxane synthase inhibitor ozagrel (100 μM), aspirin (100 μM) or theMDA scavengers, SA (1.5 mM) or 3-MoSA (1.5 mM). Next, arachidonic acid(5 μM) was added and the platelets were incubated for an additional 2 hat room temperature. The reactions were stopped by adding indomethacin(100 μM) and the aldehyde scavenger pyridoxamine (1 mM) for 30 min at 4°C., to inhibit all residual COX-1 activity and to prevent formation ofMDA adducts on proteins during the purification process, respectively.An aliquot (5 μl) of sample was used for protein estimations using a BCAassay.

In Vivo Platelet Activation: Metabolic Syndrome and Sickle CellPatients.

Formation of MDA adducts on platelet proteins during platelet activationin vitro suggested the hypothesis that MDA-protein adducts in plateletswould be elevated in states of chronic platelet activation in vivo.Because most proteins in the anuclear platelet exhibit very littleturnover, covalent adducts of these proteins would be expected toaccumulate in states of ongoing platelet activation. Accordingly, thelevels of dilysyl-MDA crosslinks in platelets were measured in twoconditions in which there is evidence of in vivo platelet activation,metabolic syndrome (MetSyn) (8, 9) and sickle cell disease (SCD) (8, 9),and compared to the levels in platelets from healthy subjects isolatedfrom blood collected in the same conditions.

Blood was collected into a 5 ml syringe containing 0.4% sodium citrate,aspirin (100 apyrase (2 Units), carbaprostacyclin (1 and SQ 29,548 (10nM) final concentration (to prevent ex vivo platelet activation). Theblood was incubated at room temperature for 40 min prior to preparationof washed platelets and protein quantification, which was performed asdescribed above. In all instances, two aliquots of washed platelets fromthe quality control (QC) were processed in parallel. Indomethacin (100μM) and pyridoxamine (1 mM) were added prior protein digestion asdescribed above.

Preparation of QC Samples from Washed Platelets.

Human platelet concentrate obtained from the Blood Bank at VanderbiltUniversity Hospital was acidified to pH 6.4 with 0.15 M citric acid.Pyridoxamine (1 mM final concentration) and indomethacin (100 μM finalconcentration) were added to the concentrate and incubated for 30 min atroom temperature. Platelets were then centrifuged at 1,000 g for 10 min.The pellet was resuspended in suspension buffer (8.3 mM sodiumphosphate, pH 6.5, 0.109 M NaCl, and 5.5 mM glucose) at finalconcentration of 600,000 cells/μl, and 100 μl aliquots (2.5 mg ofprotein) were stored at −80° C. until used.

Analysis of Protease-Derived Lysyl-MDA-Lysyl Crosslinks (Dilysyl-MDACrosslinks) in Washed Platelets by LC/ESI/MS/MS.

Samples (around 3 mg of protein) were digested with proteases aspreviously described for lysyl-lactam adducts (7). In summary, thesamples were subsequently heated at 95° C. and allowed to cool at roomtemperature, before step digestion with pronase (1 mg) at 37° C. for 24h. Pronase was inactivated by heating samples at 95° C. for 10 min andthen cooled to room temperature. Then, 1 μl of 500 mM aminopeptidase wasadded for every 1 mg of sample protein and incubated at 37° C. for 24 h.Five nanograms of ¹³C₆-dilysyl-MDA crosslink standard were added to eachplatelet sample and dilysyl-MDA crosslinks were purified using Oasiscartridge as described above in the section for the preparation ofdilysyl-MDA Crosslink standard. The eluate was dried under nitrogen flowto a volume of 100 μl, diluted up to 1 ml with a 0.1% formic acidaqueous solution, and filtered using a 0.22 μm nylon Spin-x centrifugetube spun at 6,000 rpm for 5 min. Samples were purified by HPLC throughthe Thermo Scientific Aquasil C18 column. The fractions containing theradioactivity were pooled, concentrated in Oasis HLB cartridge, and thendried under nitrogen to a final volume of 40 μl.

Pooled HPLC fractions concentrated using Oasis were analyzed by LC/MS/MSusing an YMC OD-AQ column. Solvent A (99.9% H₂O/0.1% formic acid) andsolvent B (99.9% ACN/0.1% formic acid) were filtered through a 0.45-μmfilter prior to mixing and ultrasonically degassed after mixing. Thegradient was as follows: 0-5 min 100-0% A, 5-7 min 0% A, 7-9 min 0-100%A, 9-15 min 100% A, with a flow rate of 0.2 ml/min. The massspectrometer was operated in the positive ion mode and the electrosprayneedle was maintained at 4000 V. Nitrogen was used for both the sheathand auxiliary gas at pressures of 30 and 5 arbitrary units,respectively. The optimizing skimmer offset was set at 10, capillarytemperature was 300° C. and the tube lens voltage was 195 V. Selectedreaction monitoring of specific transition ions for the precursor ionsat m/z 329→294 (dilysyl-MDA crosslink) or m/z 341→306 (IS) at −15 eVwere used. These settings were used during all experiments, includingthe validation procedure.

An SRM chromatogram of dilysyl-MDA crosslinks from a representativeexperiment evaluating the effect of platelet activation on formation ofthe crosslinks is depicted in FIG. 2, and this demonstrates that nointerfering peaks from endogenous compounds were observed at theretention times for either the dilysyl-MDA crosslink or IS,demonstrating that our purification method removes potentiallyinterfering endogenous compounds and that the observed signals arespecific for the analytes.

Synthesis of Scavenger-MDA Adducts.

A mixture of SA or 3-MoSA and TMP (0.1 mmol each) was incubated in 1 NHCl (1 ml) for 2 h, at 37° C. The reaction was then neutralized with 10N NaOH and incubated overnight at room temperature. The scavenger-MDAadducts were extracted three times with 500 μl of ethyl acetate. Theextracts were pooled and dried down under nitrogen stream, resuspendedin 100 μl of ACN-water (1:1, v/v), vortexed, and filtered through a0.22-um spin X column. The scavenger-MDA adducts were analyzed by directinfusion on the TSQ Quantum triple quadrupole mass spectrometer equippedwith a standard electrospray ionization source.

On a larger scale, TMP (0.84 ml, 5 mmol) was stirred with 1 N HCl (10ml) at room temperature and diluted with water (90 ml). It wasneutralized with K₂HPO₄ (7 g) and treated with SA (0.9 g, 5 mmol) orMoSA (1.06 g, 5 mmol) for 4 h. The adduct was extracted with ethylacetate (4×20 ml). After removal of solvents, the adduct was purified ona column of silica with ethyl acetate as eluent; yield 30%.

Determination of the Rate of Reactivity of the Scavengers with MDA InVitro.

N^(α)-Acetyllysine with or without SA, 3-MoSA or 4-HoBA was incubated at37° C. in the presence of MDA (ratio 1:1) in 0.1 M phosphate, pH 7.4.After 4 h, 10 μl aliquots were diluted to 200 μl with solvent A andanalyzed by HPLC. Solvent A consisted to 20% methanol/0.2% formic acidand solvent B was 99.8% methanol/0.2% formic acid. The gradient was asfollows: 0-1 min 100% A, 1-7 min 100-0% A, 7-14 min 0% B. PhenomenexKinetex column at a flow rate of 0.2 ml/min and the absorbance at 280 nmwere used.

LC/MS/MS Quantification of Scavengers-MDA Adducts from Washed Platelet.

Washed platelets were activated as described above in the section “exvivo activation”. The scavenger-MDA adducts were extracted three timeswith 500 μl of ethyl acetate from 50 μl of the reaction mixture, asdescribed above. The extract was dried down, re-suspended in 100 μl ofACN-water (1:1, v/v), vortexed, and filtered through a 0.22-um spin Xcolumn. The reactions were analyzed by LC/MS/MS using the column andgradient as described above. The mass spectrometer was operated in thepositive ion mode and the spray voltage was maintained at 5000 V.Nitrogen was used for the sheath gas and auxiliary gas at pressures of30 and 5 arbitrary units, respectively. The optimized skimmer offset wasset at 10, capillary temperature was 300° C. and the tube lens voltagefor both compounds was set to 49. Selected reaction monitoring ofspecific transition ions for the precursor ions at m/z 178→106 (SA-MDAadduct) or m/z 208→136 (3-MoSA-MDA adduct) at −15 eV were used.

GC/NICUMS Quantification of Thromboxane B₂.

Serum TxB₂ (sTxB₂) was measured as an indicator of inhibition ofplatelet COX activity from blood drawn into non-citrated vacuum tubes.Serum from patients was prepared by incubating the blood at 37° C. for45 min and then centrifuged at 3,200×g for 15 min. Serum was separatedand stored at −80° C. until analysis. For ex vivo experiment, 50 μl ofwashed platelets were used. sTxB₂ was assayed by stable isotope dilutiongas chromatography/mass spectrometry (GC/MS) with selective ionmonitoring as described previously (10).

Analysis of P-Selectin Expression.

An aliquot of citrated blood (10 μl) was added to 980 μl of PBScontaining PE-conjugated 10 μl anti-CD-41a antibody and 10 μlAPC-conjugated anti-P-selectin antibody (CD62P), then incubated for 30min at room temperature in the dark. At this time, platelets were fixedby adding 100 μl of 2% formaldehyde in PBS for 45 min, and analyzed byflow cytometry as described below.

Analysis of Reticulated Platelets.

Reticulated platelets were characterized by measuring platelets positivefor staining with thiazole orange following a method described by Koikeet al. (11) and modified as follows. A PE-conjugated anti-CD-41 anantibody (BD-Pharmingen) (10 μl) was added to 980 μl of PBS containingthiazole orange at 100 ng/ml. An aliquot of whole blood (10 μl) wasadded to this tube, incubated for 30 min at room temperature in thedark, and platelets were fixed by adding 100 μl of 2% formaldehyde inPBS, for 45 min. The platelets were then analyzed by flow cytometry asdescribed below.

Flow Cytometry Analysis.

Expression of markers of platelet activation were performed by flowcytometry analysis as described by Faull et al. (12). Single colorstaining controls were included in each assay to facilitate properfluorescence compensation. Samples were analyzed on a FACS canto II (BDbiosciences) (13). Increase in mean fluorescence intensity and %positive cells was recorded. Each time point and dose were compared toan unstimulated control and data was expressed as the fold increasesover unstimulated conditions or as the absolute values according to theexperimental design.

Statistical Methods.

Statistical analysis was performed using GraphPad Prism 4.0 (GraphPadSoftware, Inc., San Diego, Calif.). Data are expressed as mean±SEM,unless specified otherwise. The statistical significance was determinedusing One-way ANOVA (Tukey's Multiple Comparison Test) or t-test(two-tailed) with Welch's correction.

Results

Formation and Characterization of Dilysyl-MDA Crosslinks.

The present inventors examined the different adducts formed by thereaction of the amino acid, lysine, with MDA derived fromtetramethoxypropane (TMP), a MDA precursor, using full scan massspectrometry. Three major covalent adducts were identified: (i)N^(ε)-propenal-lysine (m/z 201), (ii) dihydropyridine (DHP)-lysine (m/z281) and (iii) the N,N′-disubstituted 1-amino-3-iminopropenal cross-linkformed by reaction of MDA with two lysines (dilysyl-MDA crosslink; m/z329). Because both the N^(ε)-propenal-lysine and the DHP-lysine adductsstill contain aldehyde moieties, they can further react with free aminesduring sample processing. The present inventors analyzed the DHP lysyladduct by mass spectrometry before and after incubation with thealdehyde scavenger, pyridoxamine. The data show that the DHP lysyladduct still reacts with free amines, causing its disappearance and theconcomitant appearance of the dipyridoxamine adduct, making themunsuitable for quantitation. In contrast, the dilysyl-MDA crosslink isno longer reactive and thus was chosen for quantification.

The full mass spectra for the dilysyl-MDA crosslink and its internalstandard (IS) adduct revealed predominant peaks at m/z 329 and m/z 341respectively as protonated molecular ions ([M+H]⁺) in LC/MS/MS. Theseprecursor ions were fragmented and the product ion spectra wereobtained. The corresponding fragments in the CID spectra obtained at −15eV and −28 eV is shown in FIG. 1. The probable cleavage reactions ofdilysyl-MDA crosslink (m/z 329) are presented in FIG. 1. The ionfragment at m/z 312, corresponding to the deaminated dilysyl-MDAcrosslink is seen at relatively low voltages, followed by dehydration toproduce m/z 294. A second dehydration from m/z 294 will produce theproduct ion m/z 276. The ion m/z 183 corresponds to the loss of onelysine from m/z 294, while m/z 129 is the cyclization of the lysinefragment (FIG. 1). The fragmentation of the IS (at m/z 341) areindicated in parenthesis (FIG. 1).

To ascertain that the fragmentation pattern observed for the dilysyl-MDAcrosslink is different from the other MDA adduct structures, we purifiedboth the N^(ε)-propenal-lysine and the DHP-lysine adducts and analyzedthem by LC/ESI/MS/MS.

Selected reaction monitoring (SRM) chromatograms for the transitions ofm/z 329 to m/z 294 and 129 (dilysyl-MDA crosslinks), and the transitionsof m/z 341 to m/z 306 and 135 ([¹³C₁₂] dilysyl-MDA crosslinks IS) areshown in FIG. 2. The fragment ions of the [¹³C₁₂] dilysyl-MDA crosslinkinternal standard elute at a similar but slightly earlier time than theunlabeled dilysyl-MDA-crosslink. The use of an initial solid phaseextraction cartridge followed by High Pressure Liquid Chromatographic(HPLC) purification minimized impurities in the sample extracts thatcould produce ion suppression of the target analytes. The use of SRMprovided high selectivity, sensitivity and intensity for bothdilysyl-MDA crosslink and IS.

Validation of LC/MS/MS Method to Measure Dilysyl-MDA Crosslink.

Assays were validated according to the U.S. FDA guidance onbioanalytical method validation (5). The measured area under the SRMcurve for the dilysyl-MDA crosslink was linear with the amount ofweighed dilysyl-MDA crosslink added to the assay. The limit of detection(LOD) and limit of quantification (LOQ) were 2.1 pg and 7.1 pg, (S/N>3).These low values are indicative of the high sensitivity of the method.The overall variability of the digestion steps was 4.0%. The intra- andinter-day precisions were acceptable with relative standard deviation of6.7 and 6.6% respectively suggesting that the method is reproducible forquantification of dilysyl-MDA crosslink in washed platelets.

Platelet Activation Leads to Modification of Platelet Proteins by MDA.

To determine whether platelet activation leads to modification ofplatelet proteins by MDA, we compared the level of protease-deriveddilysyl-MDA crosslinks in unactivated washed platelets with washedplatelets activated with 5 μM arachidonic acid for 2 h. Dilysyl-MDAcrosslinks increased from a mean of 14.2 (SE±1.2) to 43.1 (SE±5.6)following platelet activation (p<0.0001) (FIG. 3). To assess whether thecrosslinks come from proteins or from the pool of lysine available inplatelets, we compared the levels of dilysyl-MDA crosslinks from samplesdigested to single amino acids to those of samples where the lastprotease (aminopeptidase) was omitted. Our data showed that 91% of thecrosslinks are derived from adducted proteins.

Inhibitors of Thromboxane Synthesis and Scavengers of ReactiveDicarbonyls Protect Proteins from Adduction by MDA in ActivatedPlatelets.

MDA can be synthesized in platelets by sequential metabolism ofarachidonic via COX-1 followed by the thromboxane synthase (1). Toascertain that dilysyl-MDA crosslinks were formed during ex vivoplatelet activation via the COX-1-initiated thromboxane pathway, humanwashed platelets were preincubated with vehicle, the COX-1 inhibitoraspirin or the thromboxane synthase inhibitor ozagrel prior addition of5 arachidonic. Both aspirin and ozagrel reduced levels of thedilysysl-MDA crosslink to baseline levels (FIG. 3A). Together, thesedata demonstrate that MDA is generated in human platelets in aCOX-1/thromboxane synthase-dependent fashion and forms covalent adductswith proteins.

The present inventors have previously developed small molecules thatreact with the γ-ketoaldehyde levuglandins 3 orders of magnitude fasterthan with lysine, and shown that these “scavengers” protect proteinsfrom covalent adduction by these 1,4-dicarbonyls (4). They now find thatthese scavengers also protect proteins from covalent modifications bythe 1,3-dicarbonyl, MDA. Human washed platelets were pre-incubated withthe scavengers, salicylamine (SA) or 3-methoxysalicylamine (3-MoSA),prior to activation with arachidonic acid. The data show that SA and3-MoSA decreased levels of dilysysl-MDA crosslinks to baseline levels(FIG. 3A), demonstrating that the scavengers are efficient in protectingproteins from adduction by MDA. The present inventors further evaluatedthe efficacy of other SA analogs to protect protein against adduction byMDA and found that 5-ethylsalicylamine (EtSA) and 5-methylsalicylamine(MeSA) were as effective as scavengers as was SA.

To exclude the possibility that the scavengers reduced MDA adducts byinhibiting COX-1, the level of thromboxane B₂ (TxB₂) following plateletincubation with scavengers was measured. In contrast to aspirin orozagrel, which both inhibited TxB₂ levels by 96%, neither SA nor 3-MoSAsignificantly affected TxB₂ levels (FIG. 3B), demonstrating that thereduced formation of protein adducts by these compounds is due to directscavenging of MDA and not by inhibiting COX-1.

Characterization of the Product of Reaction of Scavengers with MDA.

It was hypothesized that the scavengers prevent formation of MDA-lysineadducts by preferential formation of stable MDA-scavenger adducts, andthat the greater reactivity of the scavengers with MDA results from thepresence of 2-hydroxyl in SA and its analogues. The products of thereaction of SA and 3-MoSA with MDA were characterized by LC/MS/MS as theN-propenal adducts of the scavengers. The structures ofN-propenal-3-MoSA and N-propenal-SA adduct were confirmed by NMR. Thedata show that the propenal adducts are stabilized by a hydrogen bondbetween the carbonyl of MDA and the 2-hydroxyl group on the ring.

The formation of MDA adducts of the scavengers was compared with that ofMDA adducts of lysine, employing analysis of the products on HPLC (FIG.4). Formation of the MDA adduct of 3-MoSA was 15.1 fold greater thanthat of the MDA-lysine adduct, and the formation of the MDA adduct of SAwas 9.2 fold greater than that of the lysine adduct, indicating thestrong preferential reactivity of MDA with these 2-hydroxy-benzylaminescavengers. By contrast, 4-hydroxy-benzylamine (4-HoBA) formed an MDAadduct in an amount even less than that of lysine (0.6 fold).

The hydrogen bonding of MDA with the 2-hydroxyl of the scavengers likelyfacilitates the interaction of MDA with the amino group that initiatesformation of the adduct, accounting for the preferential reactivity ofMDA with the scavengers relative to reaction with the amino group oflysine. By contrast, the 4-hydroxyl of 4-HoBA is unable to participatein H-bonding with MDA, likely due to its remoteness from the amine.

Levels of Scavenger-MDA Adducts in Human Washed Platelets.

The formation of MDA adducts of SA and 3-MoSA in activated platelets wasanalyzed. The adducts were extracted from the platelet supernatant andanalyzed by LC/MS/MS by monitoring the transitions of m/z=208 to m/z=136and m/z=178 to m/z=106 for 3-MoSA-MDA and SA-MDA, respectively (FIG. 5).We find that inhibition of dilysyl-MDA crosslinks on platelet proteinsis associated with the concomitant appearance of the equivalentscavenger-MDA adduct (FIG. 5). These results demonstrate that theprotective effect of the scavengers is due to its reaction with MDA andformation of a stable non-reactive adduct.

Increased Formation of MDA Adducts of Platelet Proteins In Vivo inPatients with Metabolic Syndrome (MetSyn).

Analysis of dilysyl-MDA crosslinks of platelet proteins providedsignificant discrimination between the group of patients with MetSyn andhealthy subjects. Levels of dilysyl-MDA crosslinks in the platelets ofpatients with MetSyn were increased 2-fold in comparison with those ofhealthy subjects (MetSyn: 6.63±3.56 ng/mg protein; healthy subjects3.38±1.43 ng/mg protein; p=0.0002) (FIG. 6A). 55% of the patients hadvalues elevated above the normal range. The capacity of platelets toproduce thromboxane A₂ ex vivo was not different between patients withMetSyn and healthy subjects (serum TxB₂ was 265.2±74.6 ng/ml in patientswith MetSyn and 278.1±114.2 ng/ml in healthy subjects).

The increases in dilysyl-MDA crosslinks were compared with those ofsingle platelet P-selectin expression, a marker of in vivo plateletactivation. Single platelet levels of P-selectin were increasedsignificantly but to a lesser extent (1.3 fold); levels in MetSyn were4.73±2.06% and healthy subjects 3.66±0.89% (p=0.0002), with values abovethe upper limit of normal in 37.5% of patients. Levels of reticulatedplatelets, indicators of platelet turnover, were only 1.2 fold increasedin MetSyn (2.18±1.55%, when compared to healthy subjects (1.85±1.18%)and the difference was not significant.

Increased Formation of MDA Adducts of Platelet Proteins In Vivo inPatients with Sickle Cell Disease.

The levels of dilysyl-MDA crosslinks in protein from platelets ofpatients with SCD (6.52±2.67 ng/mg of protein) were 2-fold higher thanthose of healthy subjects (3.38±1.43 ng/mg of protein; p<0.0001) (FIG.6B). When patients were treated with the non-selective cyclooxygenaseinhibitor, ketorolac, during admission for vaso-occlusive crisis, thelevels of platelet dilysyl-MDA crosslinks were significantly lower(3.15±1.18 ng/mg of protein), consistent with inhibition of thebiosynthesis of MDA that is derived from COX-1. Verifying the inhibitionof platelet COX1 by ketorolac, levels of serum TxB₂ during ketorolactreatment were reduced from 219.7±183.1 ng/mL to 68.9±127.7 ng/mL.

This Example demonstrates nuclear penetration of an embodiment of thepresent invention.

FIGS. 7 and 8 show that embodiments of the present invention are activein platelets, but that inhibition of LG adducts in the nucleus is a moreselective process. Thus, 3-MOSA, which is active in the platelet isinactive in the nucleus as reflected by its lack of effect on LG adductsof histones. Pentylpyridoxamine which is an effective scavenger in theplatelet is only partially effective in the nucleus. On the other hand,5-ethylsalicylamine is an effective scavenger in the nucleus. It can beconcluded that there is good penetration of the scavenger into cells,but their access to the nuclear compartment is more selective. It is thefinding that 5-ethylsalicylamine is a nuclear penetrant scavenger thatis novel. No investigator has reported on the nuclear penetration of anyscavenger heretofore, and it is this finding of selective nuclear uptakethat is new and is the basis for considering that 5-ethylsalicylamine isan agent that can protect histones and DNA from reactive dicarbonyls.

FIG. 7 shows inhibition of formation of LG adducts in platelets. Humanplatelets were pre-incubated with 10 μM dazoxiben, a thromboxanesynthase inhibitor, and 1 mM of the specified compound or vehicle(control) for 30 min. After incubation, 20 mM arachidonic acid was addedfor 2 h, and then the proteins were precipitated and digested to singleamino acids by step-digestion with proteases. The daughter ions at m/z332.1 and 84.1 for the lactam were monitored. Values are means±SD (n=9)*P<0.001 vs. control, +P<0.001 vs. 100 μM concentration.

FIG. 8 shows that an embodiment of the present invention, EtSA, blocksLG-lysyl adduct formation on histones. (A). Scavengers were screened inRAW264.7 cells for the ability to decrease LG adduct formation onhistones. Scavengers used were glucosamine (GA), 3-methoxysalicylamine(3-MoSA), pentylpyridoxamine (PPM), and 5-ethylsalicylamine (EtSA).Cells were stimulated to express COX-2, pretreated 45 min. with 500 μMscavenger or vehicle (H₂O), and given 20 μM AA for 1 h before lysing andextracting histones. Histone proteins were analyzed by LC/EST/MS/MS forLG-lysyl lactam adduct, n=2.

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It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thesubject matter disclosed herein. Furthermore, the foregoing descriptionis for the purpose of illustration only, and not for the purpose oflimitation.

We claim:
 1. A method of reducing the occurrence of malondiadehydeprotein modification in a subject in need thereof, comprising:identifying a subject with increased platelet activation; administeringto said subject a malondiadehyde adduct formation reducing amount of acompound of the following formula:

or a pharmaceutically acceptable salt thereof; to thereby reduce saidprotein modification.
 2. The method of claim 1, wherein the compound is


3. The method of claim 1, wherein the compound is selected from thefollowing formula:

or a pharmaceutically acceptable salt thereof.